Compounds useful for inhibition of farnesyl protein transferase

ABSTRACT

Novel compounds of the formula:                    
     or a pharmaceutically acceptable salt or solvate thereof, wherein: 
     a represents N or NO − ; 
     R 1  and R 3  are the same or different and each represents halo; 
     R 2  and R 4  are the same or different and each is selected from H and halo, provided that at least one of R 2  and R 4  is H; 
     T is a substituent selected from SO 2 R or                    
     Z is O or S; 
     n is zero or an integer from 1 to 6; 
     R is alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, or N(R 5 ) 2 ; 
     R 5  is H, alkyl, aryl, heteroaryl or cycloalkyl. 
     Also disclosed are methods of inhibiting farnesyl protein transferase and methods for treating tumor cells.

BACKGROUND

WO 95/10516, published Apr. 20, 1995 discloses tricyclic compoundsuseful for inhibiting farnesyl protein transferase.

In view of the current interest in inhibitors of farnesyl proteintransferase, a welcome contribution to the art would be compounds usefulfor the inhibition of farnesyl protein transferase. Such a contributionis provided by this invention.

SUMMARY OF THE INVENTION

This invention provides compounds useful for the inhibition of farnesylprotein transferase (FPT). The compounds of this invention arerepresented by the formula:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

a represents N or NO⁻;

R¹ and R³ are the same or different and each represents halo;

R² and R⁴ are the same or different and each is selected from H andhalo, provided that at least one of R² and R⁴ is H;

T is a substituent selected from SO₂R or

Z is O or S;

n is zero or an integer from 1 to 6;

R is alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl,heterocycloalkyl, or N(R⁵)₂;

R⁵ is H, alkyl, aryl, heteroaryl or cycloalkyl.

The compounds of this invention: (i) potently inhibit farnesyl proteintransferase, but not geranylgeranyl protein transferase I, in vitro;(ii) block the phenotypic change induced by a form of transforming Raswhich is a farnesyl acceptor but not by a form of transforming Rasengineered to be a geranylgeranyl acceptor; (iii) block intracellularprocessing of Ras which is a farnesyl acceptor but not of Ras engineeredto be a geranylgeranyl acceptor; and (iv) block abnormal cell growth inculture induced by transforming Ras.

The compounds of this invention inhibit farnesyl protein transferase andthe farnesylation of the oncogene protein Ras. Thus, this inventionfurther provides a method of inhibiting farnesyl protein transferase,(e.g., ras farnesyl protein transferase) in mammals, especially humans,by the administration of an effective amount of the compounds of formula1.0. The administration of the compounds of this invention to patients,to inhibit farnesyl protein transferase, is useful in the treatment ofthe cancers described below.

This invention provides a method for inhibiting or treating the abnormalgrowth of cells, including transformed cells, by administering aneffective amount of a compound of this invention. Abnormal growth ofcells refers to cell growth independent of normal regulatory mechanisms(e.g., loss of contact inhibition). This includes the abnormal growthof: (1) tumor cells (tumors) expressing an activated Ras oncogene; (2)tumor cells in which the Ras protein is activated as a result ofoncogenic mutation in another gene; and (3) benign and malignant cellsof other proliferative diseases in which aberrant Ras activation occurs.

This invention also provides a method for inhibiting or treating tumorgrowth by administering an effective amount of the tricyclic compounds,described herein, to a mammal (e.g., a human) in need of such treatment.In particular, this invention provides a method for inhibiting ortreating the growth of tumors expressing an activated Ras oncogene bythe administration of an effective amount of the compounds of formula1.0. Examples of tumors which may be inhibited or treated include, butare not limited to, lung cancer (e.g., lung adenocarcinoma), pancreaticcancers (e.g., pancreatic carcinoma such as, for example, exocrinepancreatic carcinoma), colon cancers (e.g., colorectal carcinomas, suchas, for example, colon adenocarcinoma and colon adenoma), myeloidleukemias (for example, acute myelogenous leukemia (AML)), thyroidfollicular cancer, myelodysplastic syndrome (MDS), bladder carcinoma,epidermal carcinoma, breast cancer and prostate cancer.

It is believed that this invention also provides a method for inhibitingor treating proliferative diseases, both benign and malignant, whereinRas proteins are aberrantly activated as a result of oncogenic mutationin other genes—i.e., the Ras gene itself is not activated by mutation toan oncogenic form—with said inhibition or treatment being accomplishedby the administration of an effective amount of a compound of formula1.0 to a mammal (e.g., a human) in need of such treatment. For example,the benign proliferative disorder neurofibromatosis, or tumors in whichRas is activated due to mutation or overexpression of tyrosine kinaseoncogenes (e.g., neu, src, abl, lck, and fyn), may be inhibited ortreated by the tricyclic compounds described herein.

The compounds of formula 1.0 useful in the methods of this inventioninhibit or treat the abnormal growth of cells. Without wishing to bebound by theory, it is believed that these compounds may functionthrough the inhibition of G-protein function, such as ras p21, byblocking G-protein isoprenylation, thus making them useful in thetreatment of proliferative diseases such as tumor growth and cancer.Without wishing to be bound by theory, it is believed that thesecompounds inhibit ras farnesyl protein transferase, and thus showantiproliferative activity against ras transformed cells.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms are used as defined below unlessotherwise indicated:

MH⁺—represents the molecular ion plus hydrogen of the molecule in themass spectrum;

Et (or ET)—represents ethyl (C₂H₅);

alkyl—represents straight and branched carbon chains that contain fromone to twenty carbon atoms, preferably one to six carbon atoms;

halo—represents fluoro, chloro, bromo and iodo;

cycloalkyl—represents saturated carbocyclic rings branched or unbranchedof from 3 to 20 carbon atoms, preferably 3 to 7 carbon atoms;

heterocycloalkyl—represents a saturated, branched or unbranchedcarbocylic ring containing from 3 to 15 carbon atoms, preferably from 4to 6 carbon atoms, which carbocyclic ring is interrupted by 1 to 3hetero groups selected from —O—, —S— or —NR⁹— (wherein R⁹ can be, forexample, —C(O)N(R¹⁰)₂, —CH₂C(O)N(R¹⁰)₂, —SO₂R¹⁰, —SO₂N(R¹⁰)₂, —C(O)R¹¹,—C(O)—O—R¹¹, alkyl, aryl, aralkyl, cycloalkyl, heterocycloalkyl orheteroaryl; each R¹⁰ independently represents H, alkyl, aryl, or aralkyl(e.g., benzyl); and R¹¹ is alkyl, aryl, aralkyl, heteroaryl orheterocycloalkyl)—(suitable heterocycloalkyl groups include 2- or3-tetrahydrofuranyl, 2- or 3-tetrahydrothienyl, 2-, 3- or 4-piperidinyl,2- or 3-pyrrolidinyl, 2- or 3-piperizinyl, 2- or 4-dioxanyl, etc.—withpreferred heterocycloalkyl groups being 2-, 3- or 4-piperidinylsubstituted with R¹⁰ on the piperidinyl nitrogen);

aryl (including the aryl portion of aryloxy and aralkyl)—represents acarbocyclic group containing from 6 to 15 carbon atoms and having atleast one aromatic ring (e.g., aryl is a phenyl ring), with allavailable substitutable carbon atoms of the carbocyclic group beingintended as possible points of attachment, said carbocyclic group beingoptionally substituted (e.g., 1 to 3) with one or more of halo, alkyl,hydroxy, alkoxy, phenoxy, CF₃, amino, alkylamino, dialkylamino, —COOR¹¹or —NO₂ (wherein R¹¹ is H, alkyl, aryl, heteroaryl or cycloalkyl); and

heteroaryl—represents cyclic groups, optionally substituted with R³ andR⁴, having at least one heteroatom selected from O, S or N, saidheteroatom interrupting a carbocyclic ring structure and having asufficient number of delocalized pi electrons to provide aromaticcharacter, with the aromatic heterocyclic groups preferably containingfrom 2 to 14 carbon atoms, e.g., triazolyl, 2-, 3- or 4-pyridyl orpyridyl N-oxide (optionally substituted with R¹¹ as defined above),wherein pyridyl N-oxide can be represented as:

The following solvents and reagents are referred to herein by theabbreviations indicated: ethanol (EtOH); methanol (MeOH); acetic acid(HOAc or AcOH); ethyl acetate (EtOAc); N,N-dimethyl-formamide (DMF);trifluoroacetic acid (TFA); trifluoro-acetic anhydride (TFAA);1-hydroxybenzotriazole (HOBT); 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DEC); diisobutylaluminum hydride(DIBAL); and4-methylmorpholine (NMM).

The positions in the tricyclic ring system are:

Those skilled in the art will also appreciate that the S and Rstereochemistry for the C-11 position of the tricyclic ring is asfollows:

Preferred halo atoms for R¹, R², R³, and R⁴ in formula 1.0 are selectedfrom: Br, Cl or I, with Br and Cl being preferred.

Compounds of formula 1.0 include compounds of the formula:

wherein R¹ and R³ are the same or different halo and a and T are asdefined above. Preferably, for these dihalo compounds, R¹ and R³ areindependently selected from Br or Cl, and more preferably R¹ is Br andR³ is Cl.

Compounds of formula 1.0 include compounds of formulas 1.1 and 1.2:

wherein R¹, R³ and R⁴ in formula 1.1 are halo, and R¹, R² and R³ informula 1.2 are halo. Compounds of formula 1.1 are preferred.

Preferably, in formula 1.1, R¹ is Br, R³ is Cl, and R⁴ is halo. Morepreferably, in formula 1.1, R¹ is Br, R³ is Cl, and R⁴ is Br.

Preferably, in formula 1.2, R¹ is Br, R² is halo, and R³ is Cl. Morepreferably, in formula 1.1, R¹ is Br, R² is Br, and R³ is Cl.

Also, preferably, for the compounds of this invention, substituent a inRing I represents N.

T is preferably —SO₂methyl or a group

wherein R is a 3-pydinyl N-oxide, 4-pyridinyl N-oxide, 4-piperdinyl,3-piperdinyl or 3-pyrrolidinyl group, wherein the 4-piperdinyl,3-piperdinyl or 3-pyrrolidinyl groups may be substituted on thepiperindinyl or pyrrolidinyl nitrogen with a group R⁹ which can be, forexample, —C(O)N(R¹⁰)₂, —CH₂C(O)N(R¹⁰)₂, —SO₂R¹⁰, —SO₂N(R¹⁰)₂, —C(O)R¹¹,—C(O)OR¹¹, alkyl, aryl, aralkyl, cycloalkyl, heterocycloalkyl orheteroaryl; each R¹⁰ independently represents H, alkyl, aryl, or aralkyl(e.g., benzyl); and R¹¹ is alkyl, aryl, aralkyl, heteroaryl orheterocycloalkyl.

Those skilled in the art will appreciate that compounds of formula 1.0include compounds of formulas 1.3 and 1.4:

with compounds of 1.3 being preferred for compounds of formula 1.1, andwith compounds of Formula 1.4 being preferred for compounds of formula1.2.

Thus, compounds of the invention include compounds of the formulas:

Certain compounds of the invention may exist in different isomeric(e.g., enantiomers and diastereoisomers) forms. The inventioncontemplates all such isomers both in pure form and in admixture,including racemic mixtures. Enol forms are also included.

Certain compounds of formula 1.0 will be acidic in nature, e.g. thosecompounds which possess a carboxyl or phenolic hydroxyl group. Thesecompounds may form pharmaceutically acceptable salts. Examples of suchsalts may include sodium, potassium, calcium, aluminum, gold and silversalts. Also contemplated are salts formed with pharmaceuticallyacceptable amines such as ammonia, alkyl amines, hydroxyalkylamines,N-methylglucamine and the like.

Certain basic compounds of formula 1.0 also form pharmaceuticallyacceptable salts, e.g., acid addition salts. For example, thepyrido-nitrogen atoms may form salts with strong acid, while compoundshaving basic substituents such as amino groups also form salts withweaker acids. Examples of suitable acids for salt formation arehydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic,salicylic, malic, fumaric, succinic, ascorbic, maleic, methane-sulfonicand other mineral and carboxylic acids well known to those in the art.The salts are prepared by contacting the free base form with asufficient amount of the desired acid to produce a salt in theconventional manner. The free base forms may be regenerated by treatingthe salt with a suitable dilute aqueous base solution such as diluteaqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate. Thefree base forms differ from their respective salt forms somewhat incertain physical properties, such as solubility in polar solvents, butthe acid and base salts are otherwise equivalent to their respectivefree base forms for purposes of the invention.

All such acid and base salts are intended to be pharmaceuticallyacceptable salts within the scope of the invention and all acid and basesalts are considered equivalent to the free forms of the correspondingcompounds for purposes of the invention.

Intermdiates useful in the preparation of the compounds of the inventionmay be prepared according to the procedures described in WO 95/10516published Apr. 20, 1995, in WO 96/30363 published Oct. 3, 1996, in U.S.Pat. No. 5,151,423 and by the methods described below.

Compounds of the invention can be prepared according to the reaction:

In the reaction, the carboxylic acid (14.0) is coupled to the tricyclicamine (13.0) using amide bond forming conditions well known to thoseskilled in the art. The substituents are as defined for Formula 1.0. Forexample, carbodiimide coupling methods (e.g., DEC) can be used. Forexample, the carboxylic acid (14.0) can be reacted with the tricyclicamine (13.0) using DEC/HOBT/NMM in DMF at about 25° C. for a sufficientperiod of time, e.g., about 18 hours, to produce a compound of Formula1.0.

When T is SO₂R, X is halo, preferably, chloro.

The tricyclic piperadine compound is dissolved in an appropriate solventsuch as DMF of THF. A base is added such as triethylamine, and theappropriate alkylsulfonylchloride, prepared by methods known in the art,is added to the reaction mixture at 0° C. to ambient temperature withstirring. After 1-24 hours, the reaction mixture is added to water andthe product extracted with a suitable solvent such as ethylacetate. Thecrude reaction product can then be chromatographed on a silica gelcolumn.

Alkylaminosulfonamido derivatives can be prepared similarly:

wherein the R⁵ groups may be the same or different and each is asdefined above. In this reaction, the tricyclic piperadine compound isdissolved in an appropriate solvent such as DMF of THF. A base is addedsuch as triethylamine, and the appropriate alkylaminosulfonylchloride,prepared by methods known in the art, is added to the reaction mixtureat 0° C. to ambient temperature with stirring. After 1-24 hours, thereaction mixture is added to water and the product extracted with asuitable solvent such as ethylacetate. The crude reaction product canthen be chromatographed on a silica gel column.

The carboxylic acids (14.0) and the sulfonates (14.2) are generallyknown in the art or can be prepared by methods well known in theliterature.

Compounds of Formula 13.0 can be prepared from compounds of formula13.0a:

wherein R⁶ is H, alky, carboalkoxy or any other group that can beconverted into a group T. The compounds of formula 13.0a are prepared bymethods known in the art, for example, by methods disclosed in WO95/10516, in WO 96/30363 published Oct. 3, 1996, in U.S. Pat. No.5,151,423 and those described below.

The double bond in the compounds of formula 13.0a can be cleaved byoxidation, e.g., by the method in Preparative Example 3 below, to givethe ketones of formula 15.0 below:

Compounds of Formula 13.0a wherein the C-3 postion of the pyridine ringin the tricyclic structure is substituted by bromo (i.e., R¹ is Br) canalso be prepared by a procedure comprising the following steps:

(a) reacting an amide of the formula

wherein R^(5a) is hydrogen and R^(6a) is C₁-C₆ alkyl, aryl orheteroaryl; R^(5a) is C₁-C₆ alkyl, aryl or heteroaryl and R^(6a) ishydrogen; R^(5a) and R^(6a) are independently selected from the groupconsisting of C₁-C₆ alkyl and aryl; or R^(5a) and R^(6a), together withthe nitrogen to which they are attached, form a ring comprising 4 to 6carbon atoms or comprising 3 to 5 carbon atoms and one hetero moietyselected from the group consisting of —O— and —NR^(9a)—, wherein R^(9a)is H, C₁-C₆ alkyl or phenyl;

with a compound of the formula

wherein R², R³, and R⁴ are as defined above and R⁷a is Cl or Br, in thepresence of a strong base to obtain a compound of the formula

(b) reacting a compound of step (a) with POCl₃ to obtain a cyanocompound of the formula

(c) reacting the cyano compound with a piperidine derivative of theformula

wherein L is halo selected from the group consisting of Cl and Br, toobtain a ketone of the formula below:

(d)(i) cyclizing the ketone under acid conditions (e.g., aluminumchloride, triflic acid, or sulfuric acid) to obtain a compound ofFormula 13.0a wherein R⁶ is methyl, which can be cleaved to give thecompound of formula 15.0,

Methods for preparing compounds of Formula 13.0a disclosed in WO95/10516, in WO 96/30363 published Oct. 3, 1996, in U.S. Pat. No.5,151,423 and described below employ a tricyclic ketone intermediate.Such intermediates of the formula

wherein R¹, R², R³ and R⁴ are as defined above, can be prepared by thefollowing process comprising:

(a) reacting a compound of the formula

(i) with an amine of the formula NHR^(5a)R^(6a), wherein R^(5a) andR^(6a) are as defined in the process above; in the presence of apalladium catalyst and carbon monoxide to obtain an amide of theformula:

(ii) with an alcohol of the formula R^(10a)OH, wherein R^(10a) is C₁-C₆lower alkyl or C₃-C₆ cycloalkyl, in the presence of a palladium catalystand carbon monoxide to obtain the ester of the formula

followed by reacting the ester with an amine of formula NHR^(5a)R^(6a)to obtain the amide;

(b) reacting the amide with an iodo-substituted benzyl compound of theformula

wherein R², R³, R⁴ and R^(7a) are as defined above, in the presence of astrong base to obtain a compound of the formula

(c) cyclizing a compound of step (b) with a reagent of the formulaR^(8a)MgL, wherein R^(8a) is C₁-C₈ alkyl, aryl or heteroaryl and L is Bror Cl, provided that prior to cyclization, compounds wherein R^(5a) orR^(6a) is hydrogen are reacted with a suitable N-protecting group.

Compounds of Formula 1.0 wherein substituent a is NO (Ring I) can bemade from compounds of Formula 13.0a using procedures well known tothose skilled in the art. For example, the compound of Formula 13.0a canbe reacted with m-chloroperoxybenzoic acid in a suitable organicsolvent, e.g., dichloromethane (usually anhydrous) or methylenechloride, at a suitable temperature, to produce a compound of Formula13.0b

which can then be cleaved to provide a compound of formula 15.0 above.

Generally, the organic solvent solution of Formula 13.0a is cooled toabout 0° C. before the m-chloroperoxybenzoic acid is added. The reactionis then allowed to warm to room temperature during the reaction period.The desired product can be recovered by standard separation means. Forexample, the reaction mixture can be washed with an aqueous solution ofa suitable base, e.g., saturated sodium bicarbonate or NaOH (e.g., INNaOH), and then dried over anhydrous magnesium sulfate. The solutioncontaining the product can be concentrated in vacuo. The product can bepurified by standard means, e.g., by chromatography using silica gel(e.g., flash column chromatography).

Alternatively, compounds of Formula 1.0, wherein substituent a is NO,can be made from compounds of Formula 1.0, wherein substituent a is N,by the m-chloroperoxybenzoic acid oxidation procedure described above.

Those skilled in the art will appreciate that it is preferable to avoidan excess of m-chloroperoxybenzoic acid when the oxidation reaction iscarried out on the compounds of formula 13.0a. In these reactions anexcess of m-chloroperoxybenzoic can cause oxidation of the C-11 doublebond.

Compounds of formula 1.0 wherein Z is S can be prepared from compoundsof formula 1.0 wherein Z is O by treatment with a suitable sulfurtransfer reagent such as Lawsson's reagent.

Compounds of the invention having asymmetric carbons (e.g., compounds ofthe invention wherein X is CH or N have an asymmetric carbon at the C-11position of the the tricyclic ring) can be separated into enantiomers bytechniques known in the art, e.g., by chiral salt resolution or bychiral HPLC.

Compounds useful in this invention are exemplified by the followingexamples, which should not be construed to limit the scope of thedisclosure.

3,10-dibromo-8-chloro-6,11-dihydro-11-one-5H-benzo[5,6]-cycloheptyl-[1,2-b]pyridine(2gm, 4.98 mmol) was dissolved in 20 ml of dry tetrahydrofuran under adry nitrogen atmosphere. 5ml of a 1.5 molar solution ofN-methyl-piperidine-4-magnesium chloride was added and the reactionstirred for 18 hours. The reaction mixture was washed with saturatedammonium chloride, dried over magnesium sulfate, filtered and evaporatedto a brown oil which was chromatographed on silica gel using 2.5%methanol/methylene chloride as the eluent to obtain 2.11 gm, 85% of3,10-dibromo-8-chloro-6,11-dihydro-11-(1-methyl-4-piperidinyl)-5H-benzo[5,6]cycloheptyl[1,2-b]pyridin-11-ol.FABMS (M+H)=501

Combine 25.86 g (55.9 mmol) of4-(8-chloro-3-bromo-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidine-1-carboxylicacid ethyl ester and 250 mL of concentrated H₂SO₄ at −5° C., then add4.8 g (56.4 mmol) of NaNO₃ and stir for 2 hours. Pour the mixture into600 g of ice and basify with concentrated NH₄OH (aqueous). Filter themixture, wash with 300 mL of water, then extract with 500 mL of CH₂Cl₂.Wash the extract with 200 mL of water, dry over MgSO₄, then filter andconcentrate in vacuo to a residue. Chromatograph the residue (silicagel, 10% EtOAc/CH₂Cl₂) to give 24.4 g (86% yield) of the product.m.p.=165-167° C., Mass Spec.: MH⁺=506 (CI). Elemental analysis:calculated—C, 52.13; H, 4.17; N, 8.29; found—C, 52.18; H, 4.51; N, 8.16.

Combine 20 g (40.5 mmol) of the product of Step A and 200 mL ofconcentrated H₂SO₄ at 20° C., then cool the mixture to 0° C. Add 7.12 g(24.89 mmol) of 1,3-dibromo-5,5-dimethyl-hydantoin to the mixture andstir for 3 hours at 20° C. Cool to 0° C., add an additional 1.0 g (3.5mmol) of the dibromohydantoin and stir at 20° C. for 2 hours. Pour themixture into 400 g of ice, basify with concentrated NH₄OH (aqueous) at0° C., and collect the resulting solid by filtration. Wash the solidwith 300 mL of water, slurry in 200 mL of acetone and filter to provide19.79 g (85.6% yield) of the product. m.p.=236-237° C., Mass Spec.:MH⁺=584 (CI). Elemental analysis: calculated—C, 45.11; H, 3.44; N, 7.17;found—C, 44.95; H, 3.57; N, 7.16

Combine 25 g (447 mmol) of Fe filings, 10 g (90 mmol) of CaCl₂ and asuspension of 20 g (34.19 mmol) of the product of Step B in 700 mL of90:10 EtOH/water at 50° C. Heat the mixture at reflux overnight, filterthrough Celite® and wash the filter cake with 2×200 mL of hot EtOH.Combine the filtrate and washes, and concentrate in vacuo to a residue.Extract the residue with 600 mL of CH₂Cl₂, wash with 300 mL of water anddry over MgSO₄. Filter and concentrate in vacuo to a residue, thenchromatograph (silica gel, 30% EtOAc/CH₂Cl₂) to give 11.4 g (60% yield)of the product. m.p.=211-212° C., Mass Spec.: MH⁺=554 (CI). Elementalanalysis: calculated—C, 47.55; H, 3.99; N, 7.56; found—C, 47.45; H,4.31; N, 7.49.

Slowly add (in portions) 20 g (35.9 mmol) of the product of Step C to asolution of 8 g (116 mmol) of NaNO₂ in 120 mL of concentrated HCl(aqueous) at −10° C. Stir the resulting mixture at 0° C. for 2 hours,then slowly add (dropwise) 150 mL (1.44 mole) of 50% H₃PO₂ at 0° C. overa 1 hour period. Stir at 0° C. for 3 hours, then pour into 600 g of iceand basify with concentrated NH₄OH (aqueous). Extract with 2×300 mL ofCH₂Cl₂, dry the extracts over MgSO₄, then filter and concentrate invacuo to a residue. Chromatograph the residue (silica gel, 25%EtOAc/hexanes) to give 13.67 g (70% yield) of the product. m.p.=163-165°C., Mass Spec.: MH⁺=539 (CI). Elemental analysis: calculated—C, 48.97;H, 4.05; N, 5.22; found—C, 48.86; H, 3.91; N, 5.18.

Combine 6.8 g (12.59 mmol) of the product of Step D and 100 mL ofconcentrated HCl (aqueous) and stir at 85° C. overnight. Cool themixture, pour it into 300 g of ice and basify with concentrated NH₄OH(aqueous). Extract with 2×300 mL of CH₂Cl₂, then dry the extracts overMgSO₄. Filter, concentrate in vacuo to a residue, then chromatograph(silica gel, 10% MeOH/EtOAc+2% NH₄OH (aqueous)) to give 5.4 g (92%yield) of the title compound. m.p.=172-174° C., Mass Spec.: MH⁺=467(FAB). Elemental analysis: calculated—C, 48.69; H, 3.65; N, 5.97;found—C, 48.83; H, 3.80; N, 5.97

Combine 16.6 g (0.03 mole) of the product of Preparative Example 3, StepD, with a 3:1 solution of CH₃CN and water (212.65 mL CH₃CN and 70.8 mLof water) and stir the resulting slurry overnight at room temperature.Add 32.833 g (0.153 mole) of NaIO₄ and then 0.31 g (2.30 mmol) of RuO₂and stir at room temperature give 1.39 g (69% yield) of the product.(The addition of RuO is accompanied by an exothermic reaction and thetemperature climbs from 20° to 30° C.) Stir the mixture for 1.3 hrs.(temperature returned to 25° C. after about 30 min.), then filter toremove the solids and wash the solids with CH₂Cl₂. Concentrate thefiltrate in vacuo to a residue and dissolve the residue in CH₂Cl₂.Filter to remove insoluble solids and wash the solids with CH₂Cl₂. Washthe filtrate with water, concentrate to a volume of about 200 mL andwash with bleach, then with water. Extract with 6 N HCl (aqueous). Coolthe aqueous extract to 0° C. and slowly add 50% NaOH (aqueous) to adjustto pH=4 while keeping the temperature <30° C. Extract twice with CH₂Cl₂,dry over MgSO₄ and concentrate in vacuo to a residue. Slurry the residuein 20 mL of EtOH and cool to 0° C. Collect the resulting solids byfiltration and dry the solids in vacuo to give 7.95 g of the product. ¹HNMR (CDCl₃, 200 MHz): 8.7 (s, 1H); 7.85 (m, 6H); 7.5 (d, 2H); 3.45 (m,2H); 3.15 (m, 2H).

Combine 15 g (38.5 mmol) of4-(8-chloro-3-bromo-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidine-1-carboxylicacid ethyl ester and 150 mL of concentrated H₂SO₄ at −5° C., then add3.89 g (38.5 mmol) of KNO₃ and stir for 4 hours. Pour the mixture into 3L of ice and basify with 50% NaOH (aqueous). Extract with CH₂Cl₂, dryover MgSO₄, then filter and concentrate in vacuo to a residue.Recrystallize the residue from acetone to give 6.69 g of the product. ¹HNMR (CDCl₃, 200 MHz): 8.5 (s, 1H); 7.75 (s, 1H); 7.6 (s, 1H); 7.35 (s,1H); 4.15 (q, 2H); 3.8 (m, 2H); 3.5-3.1 (m, 4H); 3.0-2.8 (m, 2H);2.6-2.2 (m, 4H); 1.25 (t, 3H).

Combine 6.69 g (13.1 mmol) of the product of Step A and 100 mL of 85%EtOH/water, then add 0.66 g (5.9 mmol) of CaCl₂ and 6.56 g (117.9 mmol)of Fe and heat the mixture at reflux overnight. Filter the hot reactionmixture through celite® and rinse the filter cake with hot EtOH.Concentrate the filtrate in vacuo to give 7.72 g of the product. MassSpec.: MH⁺=478.0

Combine 7.70 g of the product of Step B and 35 mL of HOAc, then add 45mL of a solution of Br₂ in HOAc and stir the mixture at room temperatureovernight. Add 300 mL of 1 N NaOH (aqueous), then 75 mL of 50% NaOH(aqueous) and extract with EtOAc. Dry the extract over MgSO⁴ andconcentrate in vacuo to a residue. Chromatograph the residue (silicagel, 20%-30% EtOAc/hexane) to give 3.47 g of the product (along withanother 1.28 g of partially purified product). Mass Spec.: MH⁺=555.9. ¹HNMR (CDCl₃, 300 MHz): 8.5 (s, 1H); 7.5 (s, 1H); 7.15 (s, 1H); 4.5 (s,2H); 4.15 (m, 3H); 3.8 (br s, 2H); 3.4-3.1 (m, 4H); 9-2.75 (m, 1H);2.7-2.5 (m, 2H); 2.4-2.2 (m, 2H); 1.25 (m, 3H).

Combine 0.557 g (5.4 mmol) of t-butylnitrite and 3 mL of DMF, and heatthe mixture at to 60°-70° C. Slowly add (dropwise) a mixture of 2.00 g(3.6 mmol) of the product of Step C and 4 mL of DMF, then cool themixture to room temperature. Add another 0.64 mL of t-butylnitrite at40° C. and reheat the mixture to 60°-70° C. for 0.5 hrs. Cool to roomtemperature and pour the mixture into 150 mL of water. Extract withCH₂Cl₂, dry the extract over MgSO₄ and concentrate in vacuo to aresidue. Chromatograph the residue (silica gel, 10%-20% EtOAc/hexane) togive 0.74 g of the product. Mass Spec.: MH⁺=541.0. ¹H NMR (CDCl3, 200MHz): 8.52 (s, 1H); 7.5 (d, 2H); 7.2 (s, 1H); 4.15 (q, 2H); 3.9-3.7 (m,2H); 3.5-3.1 (m, 4H); 3.0-2.5 (m, 2H); 2.4-2.2 (m, 2H); 2.1-1.9 (m, 2H);1.26 (t, 3H).

Combine 0.70 g (1.4 mmol) of the product of Step D and 8 mL ofconcentrated HCl (aqueous) and heat the mixture at reflux overnight. Add30 mL of 1 N NaOH (aqueous), then 5 mL of 50% NaOH (aqueous) and extractwith CH₂Cl₂. Dry the extract over MgSO₄ and concentrate in vacuo to give0.59 g of the title compound. Mass Spec.: M⁺⁼468.7. m.p.=123.9°-124.2°C.

The title compound can be cleaved by the methodology of PreparativeExample 3 to prepare the corresponding 11-ketone having3,10-dibromo-8-chloro substituents.

Combine 40.0 g (0.124 mole) of the starting ketone and 200 mL of H₂SO₄and cool to 0° C. Slowly add 13.78 g (0.136 mole) of KNO₃ over a periodof 1.5 hrs., then warm to room temperature and stir overnight. Work upthe reaction using substantially the same procedure as described forPreparative Example 2, Step A. Chromatograph (silica gel, 20%, 30%, 40%,50% EtOAc/hexane, then 100% EtOAc) to give 28 g of the 9-nitro product,along with a smaller quantity of the 7-nitro product and 19 g of amixture of the 7-nitro and 9-nitro compounds.

React 28 g (76.2 mmol) of the 9-nitro product of Step A, 400 mL of 85%EtOH/water, 3.8 g (34.3 mmol) of CaCl₂ and 38.28 g (0.685 mole) of Feusing substantially the same procedure as described for PreparativeExample 2, Step C, to give 24 g of the product

Combine 13 g (38.5 mmol) of the product of Step B, 140 mL of HOAc andslowly add a solution of 2.95 mL (57.8 mmol) of Br₂ in 10 mL of HOAcover a period of 20 min. Stir the reaction mixture at room temperature,then concentrate in vacuo to a residue. Add CH₂Cl₂ and water, thenadjust to pH=8-9 with 50% NaOH (aqueous). Wash the organic phase withwater, then brine and dry over Na₂SO₄. Concentrate in vacuo to give 11.3g of the product.

Cool 100 mL of concentrated HCl (aqueous) to 0° C., then add 5.61 g(81.4 mmol) of NaNO₂ and stir for 10 min. Slowly add (in portions) 11.3g (27.1 mmol) of the product of Step C and stir the mixture at 0°-3° C.for 2.25 hrs. Slowly add (dropwise) 180 mL of 50% H₃PO₂ (aqueous) andallow the mixture to stand at 0° C. overnight. Slowly add (dropwise) 150mL of 50% NaOH over 30 min., to adjust to pH=9, then extract withCH₂Cl₂. Wash the extract with water, then brine and dry over Na₂SO₄.Concentrate in vacuo to a residue and chromatograph (silica gel, 2%EtOAc/CH₂Cl₂) to give 8.6 g of the product.

EXAMPLE 1

The compound from Preparative Example 1 (0.95 gm, 1.9 mmol) wasdissolved in 34 ml of toluene. Triethylamine (1 ml) andethylchloroformate (1.82 ml, 10 eq.) were added and the reaction mixturerefluxed for two hours. The reaction mixture was cooled to ambienttemperature and evaporated to an oil. The oil was chromatographed onsilica gel using 15 to 20% ethylacetate/hexanes to obtain 0.77 gm ofethyl4-(3,10-dibromo-8-chloro-6,11-dihydro-11-hydroxy-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)-1-piperidinecarboxylate.FABMS (M+H)=559

The compound from Preparative Example 2 (0.37 gm) was dissolved in 5 mlof concentrated hydrochloric acid and refluxed for 18 hours. The mixturewas evaporated to a brown solid of the compound4-(3,10-dibromo-8-chloro-6,11-dihydro-11-hydroxy-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)-1-piperidineand used without chromatography.

The compound of Preparative Example 3 (100 mg, 0.206 mmol) was dissolvedin 2 ml of N,N-dimethylformamide. 4-Pyridylacetic acid-N-oxide (126mg,0.82mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(DEC) (0.079 mg, 0.5 mmol)), 1-hydroxybenzotriazole (HOBt) (0.056 gm,0.5 mmol), and N-methylmorpholine (0.23 ml, 2.0 mmol) were added and thereaction mixture stirred at ambient temperature. After 24 hours, thereaction mixture was added to brine and extracted with 3×15 ml ofethylacetate. The combined ethylacetate washes were combined and thesolvent evaporated under vacuo to give a gum. The gum waschromatographed flash silica gel using 10% methanol/methylenechloride asthe eluent to obtain 0.076 gm of4-(3,10-dibromo-8-chloro-6,11-dihydro-11-hydroxy-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)-1-(4-pyridinylacetyl)piperidineN1-oxide. FABMS (M+H)=622

EXAMPLE 2

The procedure of Example 1 above was followed replacing 4-pyridylaceticacid-N-oxide with N-Boc-4-piperadineacetic acid to obtain4-(3,10-dibromo-8-chloro-6,11-dihydro-11-hydroxy-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)-1-(N-BOC-4-piperdinylacetyl)piperidinein 85% yield. High resolution MS: observed=712.0975

The compound of Step A above (0.21 gm) was dissolved in 50%trifluoroacetic acid/methylenechloride and stirred for 1 hour. Thereaction mixture was evaporated to obtain an oil which was dissolved in2 ml of methylenechloride to provide4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11-hydroxy-5H-benzo[5,6]-cyclohepta[1,2-b]pyridin-11-yl)-1-piperdinyl]-2-oxoethyl]-1-piperidine.

Trimethylsilylisocyanate (234 ul, 1.47 mmol) was added and the reactionmixture stirred at ambient temperature for 15 hours. The solvent wasevaporated and the crude product chromatographed on silica gel using7.5% methanol/methylenechloride to obtain 100 mg, 62% of4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11-hydroxy-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)-1-piperdinyl]-2-oxoethyl]-1-piperidinecarboxamide.High resolution MS: observed=655.0509

EXAMPLE 3

The compound of Example 2 Step A above (0.069 g, 0.113 mmol) wasdissolved in 2 ml of N,N-dimethylformamide. Sodium carbonate (0.036 g,0.34 mmol) and bromoacetamide (0.023 g, 0.17 mmol) were added and thereaction mixture stirred at ambient temperature for 24 hours. Themixture was added to brine and extracted with ethylacetate. Theethylacetate layer was dried over magnesium sulfate, filtered andevaporated to obtain a crude solid. The crude solid was chromatographedon silica gel using 5% methanol/methylenechloride to obtain4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11-hydroxy-5H-benzo[5,6]cyclohepta-[1,2-B]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl]-1-piperidineacetamide.High resolution MS: observed=669.0666

The following compounds can be prepared utilizing the procedure ofExample 1 above and substituting the following carboxylic acids for4-pyridylacetic acid-N-oxide.

Carboxylic Acid T = in Formula 1.0 HOOCCH₂CONH₂ —OCCH₂CONH₂

HOOCCH₂CO₂H —OCCH₂CO₂H

X = O, SO2, S, or SMe X = O, SO2, S, or SMe

R = CONH2, COCH3 R = CONH2, COCH3

R¹ = CONH2, COCH3 R¹ = CONH2, COCH3 HOOCCH₂NH₂ —OCCH₂NH₂

HOOCCH₂—COOH —OCCH₂—COOH

Assays

FPT IC₅₀ (inhibition of farnesyl protein transferase, in vitro enzymeassay) and COS Cell IC₅₀ (Cell-Based Assay) were determined followingthe assay procedures described in WO 95/10516, published Apr. 20, 1995.GGPT IC50 (inhibition of geranylgeranyl protein transferase, in vitroenzyme assay), Cell Mat Assay, and anti-tumor activity (in vivoanti-tumor studies) could be determined by the assay proceduresdescribed in WO 95/10516. The disclosure of WO 95/10516 is incorporatedherein by reference thereto.

Additional assays can be carried out by following essentially the sameprocedure as described above, but with substitution of alternativeindicator tumor cell lines in place of the T24-BAG cells. The assays canbe conducted using either DLD-1-BAG human colon carcinoma cellsexpressing an activated K-ras gene or SW620-BAG human colon carcinomacells expressing an activated K-ras gene. Using other tumor cell linesknown in the art, the activity of the compounds of this inventionagainst other types of cancer cells could be demonstrated.

Soft Agar Assay:

Anchorage-independent growth is a characteristic of tumorigenic celllines. Human tumor cells are suspended in growth medium containing 0.3%agarose and an indicated concentration of a farnesyl transferaseinhibitor. The solution is overlayed onto growth medium solidified with0.6% agarose containing the same concentration of farnesyl transferaseinhibitor as the top layer. After the top layer is solidified, platesare incubated for 10-16 days at 37° C. under 5% CO₂ to allow colonyoutgrowth. After incubation, the colonies are stained by overlaying theagar with a solution of MTT(3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide,Thiazolyl blue) (1 mg/mL in PBS). Colonies can be counted and the IC₅₀'scan be determined.

Results from the in vitro enzyme assay are given in Table 1.

TABLE 1 Example No. Compound FPT IC50 (μM) #1 Step C

<0.002 #2 Step C

0.0108 #2 Step A

0.042 #3

0.0087 #2 Step B

0.0125

For preparing pharmaceutical compositions from the compounds describedby this invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets and suppositories. Thepowders and tablets may be comprised of from about 5 to about 70 percentactive ingredient. Suitable solid carriers are known in the art, e.g.magnesium carbonate, magnesium stearate, talc, sugar, lactose. Tablets,powders, cachets and capsules can be used as solid dosage forms suitablefor oral administration.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides or cocoa butter is first melted, and the activeingredient is dispersed homogeneously therein as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool and thereby solidify.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection.

Liquid form preparations may also include solutions for intranasaladministration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally.The transdermal compositions can take the form of creams, lotions,aerosols and/or emulsions and can be included in a transdermal patch ofthe matrix or reservoir type as are conventional in the art for thispurpose.

Preferably the compound is administered orally.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component, e.g., an effectiveamount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from about 0.1 mg to 1000 mg, more preferably fromabout 1 mg. to 300 mg, according to the particular application.

The actual dosage employed may be varied depending upon the requirementsof the patient and the severity of the condition being treated.Determination of the proper dosage for a particular situation is withinthe skill of the art. Generally, treatment is initiated with smallerdosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day if desired.

The amount and frequency of administration of the compounds of theinvention and the pharmaceutically acceptable salts thereof will beregulated according to the judgment of the attending clinicianconsidering such factors as age, condition and size of the patient aswell as severity of the symptoms being treated. A typical recommendeddosage regimen is oral administration of from 10 mg to 2000 mg/daypreferably 10 to 1000 mg/day, in two to four divided doses to blocktumor growth. The compounds are non-toxic when administered within thisdosage range.

The following are examples of pharmaceutical dosage forms which containa compound of the invention. The scope of the invention in itspharmaceutical composition aspect is not to be limited by the examplesprovided.

Tablets No. Ingredients mg/tablet mg/tablet 1. Active compound 100 5002. Lactose USP 122 113 3. Corn Starch, Food Grade,  30  40 as a 10%paste in Purified Water 4. Corn Starch, Food Grade  45  40 5. MagnesiumStearate  3  7 Total 300 700

Method of Manufacture

Mix Item Nos. 1 and 2 in a suitable mixer for 10-15 minutes. Granulatethe mixture with Item No. 3. Mill the damp granules through a coarsescreen (e.g., ¼″, 0.63 cm) if necessary. Dry the damp granules. Screenthe dried granules if necessary and mix with Item No. 4 and mix for10-15 minutes. Add Item No. 5 and mix for 1-3 minutes. Compress themixture to appropriate size and weigh on a suitable tablet machine.

EXAMPLE B

Capsules No. Ingredient mg/capsule mg/capsule 1. Active compound 100 5002. Lactose USP 106 123 3. Corn Starch, Food Grade  40  70 4. MagnesiumStearate NF  7  7 Total 253 700

Method of Manufacture

Mix Item Nos. 1, 2 and 3 in a suitable blender for 10-15 minutes. AddItem No. 4 and mix for 1-3 minutes. Fill the mixture into suitabletwo-piece hard gelatin capsules on a suitable encapsulating machine.

While the present invention has been described in conjunction with thespecific embodiments set forth above, many alternatives, modificationsand variations thereof will be apparent to those of ordinary skill inthe art. All such alternatives, modifications and variations areintended to fall within the spirit and scope of the present invention.

What is claimed is:
 1. A compound of the formula:

or a pharmaceutically acceptable salt or solvate thereof, wherein: arepresents N or NO^(−;) R¹ is bromo and R³ is chloro; R² and R⁴ are thesame or different and each is selected from H and halo, provided that atleast one of R² and R⁴ is H; T is

wherein: Z is O; n is an integer from 1 to 6; and R is a 3-pyridinylN-oxide or 4-pyridinyl N-oxide.
 2. The compound of claim 1 having theformula:

wherein a, T, R¹ and R³ are as defined in claim
 1. 3. The compound ofclaim 1 having the formula:

wherein a, T, R¹, R³ and R⁴ are as defined in claim
 1. 4. The compoundof claim 1 having the formula:

wherein a, T, R¹, R² and R⁴ are as defined in claim
 1. 5. The compoundof claim 1 having the formula:

wherein a, T, R¹, R², R³ and R⁴ are as defined in claim
 1. 6. Thecompound of claim 1 having the formula:

wherein a, T, R¹, R², R³ and R⁴ are as defined in claim
 1. 7. Thecompound of claim 3, wherein R⁴ is bromo.
 8. The compound of claim 4,wherein R³ is chloro.
 9. The compound of claim 7, wherein the carbon inthe C-11 position is in the R-configuration.
 10. The compound of claim8, wherein the carbon in the C-11 position is in the R-configuration.11. The compound of claim 7, wherein the carbon in the C-11 position isin the S-configuration.
 12. The compound of claim 8, wherein the carbonin the C-11 position is in the S-configuration.
 13. A compound havingthe formula:


14. A pharmaceutical composition for inhibiting farnesyl proteintransferase comprising an effective amount of compound of claim 1 incombination with a pharmaceutically acceptable carrier.